Protocol Guide: Cortical GABAergic Neuron Monoculture

What are GABAergic Neurons?

Located in the basal forebrain, GABA neurons contain the neurotransmitter gamma-aminobutyric acid (GABA). GABA is an amino acid and the primary inhibitory neurotransmitter for the central nervous system, also known as the CNS. Because GABA reduces neuronal excitability and glutamate increases neuronal excitability, the balance between GABA and glutamate is important for proper neurologic function. Cortical GABAergic neurons can be used to study and understand diseases such as epilepsy, Alzheimer’s disease, and schizophrenia.

We provide, through partnership with BrainXell, fully differentiated cortical GABAergic neurons that express GABA and MAP2 at DIV7 with synchronous firing by DIV21 and release as the primary neurotransmitter GABA. Here we describe a protocol for seeding and culturing cortical GABAergic neurons in monoculture.

Section Overview

• Cortical GABAergic Neuron Culture Materials
• Cortical GABAergic Neuron Culture Protocol
• Cortical GABAergic Neuron Culture Related Products

Cortical GABAergic Neuron Culture Materials

Neuron cryovials must be stored immediately a liquid or vape nitrogen storage system. Store BrainFast supplements at -80°C for up to 18 months or -20°C for up to 6 months. Cryovial should be returned to -20°C between each use to maintain neuron stability.

• BrainXell Human Cortical GABAergic Neurons (BX-0400-30-1M)
• BrainFast GABA Seeding Supplement (BX-2400-100uL)
• BrainFast D4/Day 4 Supplement (BX-2040-100uL)
• BrainFast SK/Supplement K (BX-2020-100uL)
• DMEM/F12 Medium, +L-glutamine, +HEPES (D0697)
• Neurobasal Medium (Thermo Fisher Scientific #21103049)
• B-27 Supplement (Thermo Fisher Scientific #17504044)
• N-2 Supplement (Thermo Fisher Scientific #17502048)
• GlutaMAX Supplement (Thermo Fisher Scientific #35050061)
• Cultrex (R&D Systems #3432-001-01)
• Ascorbic Acid (A8960)
• PDL-coated 96-Well Plate (M0812, LPDL001)

Cortical GABAergic Neuron Culture Protocol

Day 0: Seeding Preparation

You will need to seed 4.4 - 5.5 million live cells for a 96-well plate, but additional cells can be used depending on experimental needs. For further viability and seeding information, see the Certificate of Analysis(CoA).

1. Make the Basal Medium from the components listed below in the cell culture hood or biological safety cabinet. Do not place the medium in a 37°C water bath; allow medium to come to room temperature for 15 minutes. Store at 4°C for up to 3 weeks.
   1. The addition of growth factors is optional – recommended final concentrations are: 10µg/ml GDNF, 2µg/ml TGF-β1, and 10µg/ml BDNF.

2. Add 495µl of cold DMEM/F12 medium to a 55µl aliquot of frozen Cultrex taken directly from the -80°C freezer. Mix solution to dissolve. Store at 4°C.
3. Prepare solutions for cell seeding by adding 3ml of Basal Medium to a 50ml conical tube and 25µl of Trypan Blue solution to a separate microcentrifuge tube for cell counting.

Day 0: Seeding the GABAergic Neurons

1. Place a cryovial of GABAergic neurons in a 37°C water bath to thaw. Be sure to not submerge the cap to minimize contamination.
2. When the last of the ice melts remove the vial from the water bath and disinfect with 70% ethanol. Transfer to the cell culture hood.
3. Using a P1000 pipette at a rate of 2-3 drops/sec, transfer 500µl of the Basal Medium from the 50ml conical tube to the GABAergic neuron vial; this transfer should take about 10 seconds.
4. Transfer the 1ml cryovial contents to the 50ml conical tube.
5. Centrifuge the cells at 465xg for 5 minutes.
6. Aspirate the supernatant and resuspend the cell pellet in 950µl fresh Basal Medium by using a P1000 pipette to gently pipette up and down.
7. Resuspend the cells to 1.0 x 10⁶ live cells/ml final concentration based on the CoA. Slowly add Basal Medium to the existing ~1ml in the conical tube to get to the desired concentration.
   1. For example, 5.3 million viable cells/vial is diluted to 5.3 final volume.
8. Count the cells using the Trypan Blue solution (Step 3). Pipette up and down 3-5 times to evenly suspend cells in the Basal Medium. Transfer 25µl of the cell suspension to the microcentrifuge tube filled with 25µl Trypan Blue solution and mix by pipetting. Count the number of viable and dead cells using a hemocytometer and determine the live cell concentration and viability.
9. Calculate the volume needed to create a GABAergic neuron seeding suspension. The typical seeding density is 40,000-50,000 viable cells/100µl/well for a 96-well plate (~125,000-156,250 viable cells/cm²), but the recommended seeding density may vary (refer to the CoA for lot-specific information). Based on the Trypan Blue cell count above, dilute the cells to the desired seeding concentration.

Example dilution calculation:

10. In a new 50ml conical tube, mix the previously calculated volumes of the GABAergic neurons and the Basal Medium to obtain 11ml of the GABAergic neuron seeding suspension.
11. Make the Seeding Medium for the GABAergic neurons:

12. Transfer the final Seeding Medium to a PDL-coated 96-well plate at 100µl/well after mixing completely. Do NOT move the plate during the seeding process, as movement can lead to uneven attachment.
13. Rest plate after seeding to allow the cells to settle for 10 minutes. Transfer plate to the humidified incubator at 37°C with 5% CO₂.

This protocol should not exceed 1 hour; the post-thaw health and viability of the GABAerguic neurons can be impacted if the process takes too long.

Day 4: GABAergic Neuron Medium Replacement

1. Prepare Day 4 Medium:

2. Add 100µl Day 4 Medium per well for a total volume of 200µl medium/well.

Day 7 and Onward: GABAergic Neuron Medium Changes

1. Using the Basal Medium, change half of the media weekly, i.e. on Day 7, 14, 21, etc. Remove 100µl/well and add 100µl/well of Basal Medium slowly to the entire plate.
   1. NOTE: Adding low concentrations of BrainFast SK (0.1-0.5X) in the medium may be helpful for long-duration cultures.

GABAergic neurons mature rapidly. They can be maintained in culture under the above conditions for at least 3 weeks post-seeding.

*These products are available in only the US and UK.